Francisella tularensis, the aetiological agent of tularemia, is one of the most infectious bacterial pathogens currently known and a NIAID category A priority pathogen because of its potential for use as a biological weapon. In F. tularensis, the SspA protein family members MglA and SspA play a critical role in regulating the expression of virulence genes that are essential for intramacrophage growth and survival, and are key to the organisms pathogenic lifestyle. MglA and SspA form a complex that associates with RNA polymerase (RNAP) to positively control the expression of virulence genes by a mechanism that is not understood. We have found that in the live vaccine strain of F. tularensis, the MglA-SspA complex works in concert with the small molecule ppGpp, and a putative DNA-binding protein we have named PigR, to control the expression of a common set of genes. We have obtained evidence that PigR interacts directly with the MglA-SspA complex, and that ppGpp exerts its effect by promoting this interaction. Although ppGpp plays a critical regulatory role in many other pathogens, our findings suggest that in Francisella ppGpp may control gene expression through a novel mechanism-by promoting the interaction between a transcription activator and an RNAP-associated complex formed by members of the SspA protein family. In Aim 1 of this proposal we will use a combination of biochemical and genetic approaches to determine how MglA, SspA, PigR and ppGpp exert their transcriptional effects. In particular, we propose to determine whether PigR functions through contact with the DNA, to explicitly test whether the protein-protein interaction between PigR and the MglA-SspA complex is key to the regulatory activities of these three proteins, and to determine how ppGpp modulates the interaction between PigR and the MglA-SspA complex. Our recent findings implicate the TrmE protein in the control of MglA/SspA-regulated genes. TrmE is a putative GTPase that has been shown to influence protein synthesis in other organisms. We hypothesize that TrmE exerts its effects on gene expression through an effect on the intracellular concentration of ppGpp, MglA, SspA, PigR, or another regulator. In Aim 2, we will take both genetic and biochemical approaches to determine how TrmE influences the expression of MglA/SspA-regulated genes. The proposed work is expected to reveal how an important intracellular pathogen regulates the expression of genes required for survival in the host, and has implications for how ppGpp and SspA family members regulate gene expression in other pathogenic bacteria.